Figure 10. Attenuated ATR IR spectroscopy used to probe the electronic connection between the hydrogenase and NAD⁺-reducing enzyme moiety on carbon particles.

(A) Main inactive and active states of the catalytic site of E. coli Hyd1, together with the v_CO wavenumber positions observed for each state. B: indicates basic site(s) that accept protons during catalysis, which could include co-ordinated cysteine sulphur atoms, and other surrounding amino acid residues. (B) Schematic representation of the experimental set-up for testing particles modified with a mixture of hydrogenase and NAD⁺-reducing moiety. (C) Set of ATR IR spectra recorded during solution exchange experiments. The cell solution contained 50 mM Tris–HCl (pH 8.0), and the H₂/N₂ and NAD⁺/NADH concentrations were varied as indicated on the figure. (D) Schematic representation of the experimental set-up for testing the immobilised enzymes physically separated by a piece of carbon paper (Toray TGP-H-030), in response to changes in solution H₂/N₂ or NAD⁺/NADH ratio. For these experiments, the hydrogenase was E. coli Hyd1 [18], and the NAD⁺-reducing moiety was HoxH^I64AYFU of R. eutropha soluble hydrogenase, with the hydrogenase portion inactivated by an I64A exchange in the HoxH subunit (prepared according to established protocols) [46]. Carbon particles were BP2000 carbon black.